| Background:RA is a chronic inflammatory disease characterized by systemic joint pain and destructive synovitis.Various pro-inflammatory cytokines and their corresponding signal molecules,such as lymphocytes,macrophages,TNF,IL-1,IL-6,granulocyte colony-stimulating factor,and Janus kinases are the critical pathogenesis of the disease.CD4~+Th cells play an important role in the development of RA by regulating adaptive immune responses.Th17 cells is closely related to the pathogenicity and disease activity of RA.Compared with Th17 cells,Treg cells,as a special subset of Th cells,have the opposite effect on autoimmunity and inflammation,and play a role in maintaining self-balance and self-tolerance.Therefore,the balance of Th17/Treg cells controls the development of RA and inflammatory reactions.AhR is a ligand-activated transcription factor that can play an important role in CD4~+T cell differentiation.Our previous studies suggested that smoking may activate AhR in PBMCs of RA patients and further affect the differentiation of Th17 cells,thereby participating in the pathogenesis of RA.However,we found through subgroup analysis that the expression level of AhR and its downstream target gene-CYP1A1 in non-smoking healthy individuals were higher than in non-smoking RA patients.We speculated that there may be endogenous ligands that play a protective role in rheumatoid arthritis by activating AhR and increasing its expression.Objective:(1)To screen differentially expressed metabolites between RA patients and healthy volunteers using non-targeted metabolomics techniques and identify endogenous ligands of AhR;(2)To investigate differences in gut microbiota between RA patients and healthy volunteers,and analyze the correlation between dominant or disadvantageous microbial populations and the expression of the screened metabolites;(3)To determine the effects of target metabolites on inducing the differentiation of PBMC into Th17 cells and Treg cells;(4)To reveal the effect of target metabolites on the progression of CIA in rats and its mechanism.Method:Part Ⅰ:According to the inclusion and exclusion criteria,14 non-smoking RA patients and 14 non-smoking healthy volunteers matched with age and sex of RA patients were recruited.We collected basic information and disease data of all participants to calculate DAS28,and collect whole blood and separate plasma.we employed liquid chromatography-mass spectrometry-based untargeted metabolomics to detect and analyze the plasma metabolites of the subjects,and completed the PCA and OPLS-DA,differential metabolites screenings,Metabolite set enrichment analysis and pathway analysis,and biomarker screening analysis.Part Ⅱ:The research participants and grouping schemes of this experiment are the same as partⅠ.The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified by PCR and the PCR products were purified,identified,and quantified.The Miseq library was constructed and sequenced on the Illumina platform to obtain the original data and complete quality control,OTU datasets management and analysis,taxonomic analysis,analysis of community composition,diversity analysis,differential flora screening and correlation analysis.Part Ⅲ:We collected whole blood from healthy volunteers and isolate PBMCs to observe the effect of different concentrations of IPA on PBMC activity and select the optimal stimulation concentration by CCK8 method.We detected the expression of AhR and CYP1A1 at m RNA or protein level in PBMCs after IPA stimulation by PCR or western blot.The CD3/CD28 antibodies were used to stimulate PBMCs.We observed the effects of IPA on inducing the differentiate of PBMCs into Th17 cells under the action of IL-1β,IL-6,TGF-β,anti-IFN-γand anti-IL-4 and Treg cells under the action of IL-2,TGF-β,anti-IFN-γand anti-IL-4.We also detected the proportion of Th17 cells and Treg cells,and the protein level of CYP1A1,p38,pp38,IRF4 and pIRF4.Part Ⅳ:We used SD rats to establish CIA model to verify whether IPA can improve the condition of RA.The MTX was used as the standard treatment of CIA to evaluate the IPA treatment.The effect of IPA on CIA was evaluated by arthritis score,inflammatory index and cytokines.The ratio of Th17 cells and Treg cells and the expression levels of AhR,CYP1A1 and RORγT were detected.The m RNA level of Foxp3 and the protein level of CYP1A1,p38,pp38,IRF4 and pIRF4 in spleen cells were used to explore the mechanism of IPA improving CIA.Result:Part Ⅰ:A total of 126 differential metabolites were identified in this experiment.Enrichment analysis revealed that 5 metabolic pathways were involved in the development of rheumatoid arthritis.Among them,tryptophan metabolism was the main metabolic pathway.Further analysis found that IPA had the largest difference between the two groups,and it also had the highest contribution to the grouping.Further analysis showed that IPA had a high screening value for RA and was negatively correlated with DAS28.Part Ⅱ:A total of 293 OTUs were identified in the experiment,and the dominant microbial composition of the RA group samples and the healthy control group samples was similar.Theα-analysis results showed no significant differences in microbial diversity and species richness between the two groups of samples.Theβ-analysis results indicated differences in microbial composition between the two groups.Further analysis of species differences identified differential microbes at the genus level with the top 10 abundance.According to the correlation analysis between the differential metabolites obtained in partⅠand the fecal microbiota at genus level,it was found that there were significant positive correlations between IPA and Lachnospiraceae＿NK4A136＿group and Coprococcus.Part Ⅲ:Based on the CCK8 test results,the treatment concentration of IPA was selected as 500μM and the treatment time was 72 hours.The experiment found that IPA can significantly reduce the proportion of PBMCs differentiating into Th17 cells,but this effect was partially reversed by the AhR antagonist CH223191.Under the action of IPA,the ratio of pp38/p38 and IRF4/pIRF4 were decreased,and the effect of IPA on the ratio of pp38/p38 and IRF4/pIRF4 decreased under the action of CH223191.IPA can significantly promote the differentiation of Treg cells.Compared with the Treg group,the number of PBMCs induced to differentiate into Treg cells in the IPA group increased.However,this effect was significantly weakened under the action of CH223191.Part Ⅳ:When the dose of IPA reached 20 mg/kg,it significantly reduced the progression of CIA.Compared to the CIA group,IPA treatment did not show severe changes such as joint erosion,deformation,bone erosion,and osteophyte formation.Similarly,histological examination revealed a significant decrease in synovial destruction and inflammatory cell infiltration in the CIA rats treated with IPA.During the induction of CIA,IPA increased the level of plasma IL-10 in rats and reduced the levels of plasma IL-17A,TNF-α,and IL-1β.However,the effect of IPA on CIA rats was weakened in the presence of CH223191.Conclusion:(1)The expression level of IPA in RA patients is lower than that in healthy volunteers,and IPA can be the protective factor and biomarker of RA.(2)The expression of IPA is correlated with the abundance of Lachnospiraceae＿NK4A136＿group,and the gut microbiota may influence RA by influencing tryptophan metabolites which is the endogenous ligands for AhR(3)IPA can reduce the differentiation of PBMCs into Th17 cells through AhR/p38/IRF4 signaling axis and promote the differentiation of PBMCs into Treg cells through AhR pathway;(4)IPA can alleviate collagen-induced arthritis in rats via AhR pathway. |
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